Bacterial growth, nutrition and identification Flashcards
1
Q
what are the different shapes of bacteria (4)
A
-
Coccus
- Streptococcus – causes Pneumonia
- Staphylococcus – causes Skin infections
-
Rods
- Escherichia coli – causes Gut commensal, Diarrhoea & Bloodstream infections
-
Comma shaped
- Vibrio cholerae – causes Excessive diarrhoea
-
Spirochete (spiral-shaped)
- Treponema pallidum - causes Syphilis
2
Q
how is the small size of bacteria advantageous
A
Bacteria are 10x smaller than eukaryotic cells
- This allows rapid metabolism as the diffusion of nutrients is not a limiting (as it is in eukaryotes)
- Rapid metabolism ⇒ Rapid turn-over of sugars, amino acids, nucleotides ⇒ Rapid growth
3
Q
how does bacteria grow (in numbers)
A
- Bacteria grow exponentially: 1 → 2 → 4 → 8 etc.(2n)
- Under optimal conditions: Escherichia coli divides every 20 minutes: in 11 hours from 1 to 7 billion cells
4
Q
what are the conditions required for bacteria growth
A
The Medium needs to contain: carbon, nitrogen, sulphur, phosphorus, minerals (Fe2+, Mg2+,C a2+)
5
Q
at what pH do most bacteria see optimal growth and what is the exception - why is the exception?
A
- Most pathogens exhibit optimal growth around physiological pH (7.4)
- Exceptions -Helicobacter pylori
- It is the cause of stomach ulcers
- Grows in stomach, pH 3
- The bacterium creates a micro-environment with a higher pH through production of ammonia and bicarbonate by using urea present in blood.
- So although it lives in area where pH is very acidic it raises pH to high levels using above mechanism
6
Q
what are the 2 types of bacteria
A
- aerobic
- anaerobic
7
Q
what are the 2 forms of anaerobic bacteria
A
- Aerotolerant anaerobes: can tolerate and grow in air
- Obligate anaerobes: oxygen inhibits growth or kills the cells
8
Q
are the majority of bacteria in the human gut aerobic or anaerobic
A
The majority of bacteria in the human gut are anaerobes.
9
Q
what happens when obligate anaerobes leave our bodies
A
- Many of them have survival strategy where they produce spores.
- These are very robust structures that can tolerate:
- high heat, O2 and UV radiation.
- They germinate when conditions are favourable.
10
Q
what are the 2 categories of media
A
- Media can be split into two categories:
- Selective media: Used to isolate specific bacteria by inhibiting growth of others
- Differential media: Used to distinguish between different bacteria
- Example: MacConkey Agar (both Selective and differential)
- Selective as it inhibits growth of many bacteria by the presence of bile salts and crystal violet
- Differential as the medium contains lactose.
- E. coli ferments lactose → drop in pH → pH indicator turns yellow
- Salmonella can grows on the agar but cannot ferment lactose so medium stays pink.
- This helps you distinguish between the bacteria.
11
Q
what are the different types of media that we can use to grow bacteria (2)
A
-
Liquid culture:
- Used to quantify growth rate of bacteria
- Used to study physiology of bacteria
-
Solid media (agar):
- Used to make a Preliminary identification of bacterium
- Used to Quantify number of live bacteria
- Used to Isolate a pure culture (1 colony)
12
Q
what is a defined medium
A
- Consists of pure chemicals of very specific amounts.
- These media give very reproducible growth
- Below is example of defined medium for E.coli. These media give very reproducible growth
13
Q
what is a complex medium
A
-
Complex medium:
- Made up of digests of microbial, plant and/or animal products. E.g. BHIS
- They are not defined as you don’t know exactly what compounds are present in the media.
- They allow the growth of a wide range of microbes
14
Q
what are the 4 phases of bacterial growth
A
- lag phase
- exponential phase
- stationary phase
- death phase
15
Q
describe the lag phase
A
- Not much growth
- Bacterium Adapts to new environment
- It starts up metabolism: generate ATP and make new ribosomes for protein production.
16
Q
describe the exponential phase and when it ends
A
- Rapid cell growth & metabolism
- This phase comes to an end when cells run out of one or more nutrients and/or waste products.
17
Q
describe the stationary phase
A
- Slow/no growth
- Bacteria prepare for survival under conditions of no nutrients
- Some start to sporulate – form spores which are resistance to stresses.
- Some bacteria produce antibiotics to kill neighbouring cells to allow them to grow on nutrients and macromolecules released from dead cells.
18
Q
describe the death phase
A
culture starts to die
19
Q
what is the gram-stain and what is it used for
A
- The gram stain is a rapid stain of cells by crystal violet and safranin
- It is used for rapid identification of bacteria by microscopy.
- It allows you to visualise and distinguish bacteria in two groups
20
Q
how does gram-stain distinguish between 2 groups of bacteria
A
- Gram-negative bacteria (pink)
- Gram-positive bacteria (purple)
21
Q
why is gram-stain important
A
- The gram stain is important as it Reflects fundamental differences in the bacterial cell wall.
- This is important as there are differences in the susceptibility of gram positive and gram-negative bacteria to antibiotics:
- Outer membrane of Gram-negatives bacteria functions as a barrier to many antibiotics.
- Gram positive bacteria do not have an outer membrane.
22
Q
how can a micro-biologist find a cause of infection clinically (4)
A
- There are many different approaches:
- Immunological and antigen assays
- Molecular biology assays (PCR assays, whole-genome sequencing)
- Growth-dependent microbiology
- Direct Microscopy
23
Q
A
24
Q
what do you need for direct microscopy
A
- standard light microscope
- staining
25
Q
what are the advantages of direct microscopy
A
- Very cheap, very fast
- Very useful in low-resource settings
26
Q
how are bacteria samples analysed
A
- Before we are able to figure out what bacterium is causing the infection, we first need to grow the sample on agar (gold standard)
- We then determine specific biochemical traits (e.g. growth on different carbon sources)
- Challenging:
- Not all bacteria can be grown easily
- Identification using biochemical traits not always correct, takes time
- Instead of using biochemical traits, we can analyse the sample in a Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometer.
27
Q
what is the method of MALDI-TOF (Matrix-assisted laser desorption/ionization - time of flight mass spectrometer).
A
- You spot some colony material on plate.
- These are then ionised and then accelerated into a tube.
- This separates the fragments of colony material based on mass and charge.
- This leads to peak patterns which are specific to particular species of bacteria.
- This allows you to identify which microorganism are grown on the plate.
- This takes a couple of minutes.
28
Q
there is type of pathogen which can not be cultured, name it and describe how it is analysed
A
- Treponema pallidum - causative agent of syphilis,
- You can instead Detect anti-Treponema antibodies in serum.
- These are produced by the infected individual during infection
- Generalised outline of antibody detection assays:
- You buy an assay from a manufacturer where the antigen is bound to a well and a blocking agent is added on the other binding sites.
- Then add your sample.
- If an antibody is present the antigen will bind to it.
- Then you wash away unbound sample.
- Then add secondary antibody (antihuman) which binds all antibodies coming from humans.
- This antibody has enzyme linked to it which you can then detect by adding substrate which is converted by enzyme to coloured product.
29
Q
how are antigens detected & in which condition is antigen detection used
A
- Antigens are a specific components of pathogenic bacteria. These can be detected.
- This has been proposed for the diagnosis of cerebrospinal fluid (CSF) - detect antigens of classic bacterial meningitis pathogens.
- Method:
- You use latex beads coated in antibody.
- Then add CSF sample.
- If antigens are present they bind to antibody and crosslink all the latex beads.
- Then you get aggregation which can be seen.
30
Q
describe the lateral flow test to detect antigens
A
- Another way antigens can be detected is using lateral flow tests (used for COVID-19 rapid testing).
- Method:
- Spot a sample which may contain antigen on a strip - the sample diffuses through strip.
- When the antigens reach a particular of the strip, they bind to antibodies conjugated with a label.
- If antigen is bound to the first cluster of antibodies it will also bind to a second row of specific antibodies ⇒ indicates sample is positive.
- There is also a control lane that captures any antibodies that are passing and should always show up as positive.
31
Q
what do you need to carry out PCR (4)
A
- What you need:
- Sample which may or not contain DNA of pathogen
- Primers (complementary to target DNA)
- dNTPs (nucleotides in DNA)
- polymerase (Taq) in multiple cycles (amplify DNA),
32
Q
what is the method of PCR (3 steps)
A
- 3 steps: denaturation, annealing, extension
- Method
- First denature DNA template so it becomes single stranded.
- Primers then anneal to DNA template and then polymerase along with dNTP will amplify DNA.
- With every cycle you go through you get more products formed that amplify sequence of interest.
- This is exponential amplification.
- When you have so many copies you can visualize PCR products on agarose gel in presence of dye binding to double stranded DNA.
- This will separate products based on size and this allows you identify different types of bacteria in your sample.
33
Q
what is quantitative PCR
A
- Quantitative PCR is another DNA-based methodology
- It is used to detect amplification of DNA in real time.
34
Q
what are the 2 ways used to detect DNA in quantitative PCR
A
- use dye e.g. SYBR green (this binds to double stranded DNA)
- taqman assay
- When doing PCR a probe is annealed to the part of DNA you want to amplify.
- On one side of this probe there is a fluorescent dye.
- This is switched off (quenched) because on other side of probe there is quenching molecule.
- However, when you get PCR amplification this fluorescent dye is cleaved off probe by TAQ and by itself it becomes fluorescent again.
- This allows us to quantify how much PCR products are being formed - So more amplification you have the more fluorescence signal you get
35
Q
what are the advantages of DNA-based methodologies
A
- Rapid:
- 4 – 6 hrs from clinical sample to result, whilst growing a bacterium on a plate takes 24 hrs
- Particularly useful when culture is difficult or will take a very long time
36
Q
what are the limitations + solution
A
-
Limitation
- Primers determine what you will find:
- Chlamydia in sample yes/no?
- But you cannot approach sample in completely hypothesises freeway?
- Primers determine what you will find:
-
Solution
- Sequence (‘read’) all DNA in a sample to rapidly ⇒ identify all microbes present (but not RNA viruses!)
- If you isolate DNA from sample you won’t isolating RNA so you will miss RNA virus that are present (limitation)
- Sequence (‘read’) all DNA in a sample to rapidly ⇒ identify all microbes present (but not RNA viruses!)
37
Q
how is DNA sequencing done in clinical setting
A
Outline of process:
- Take a sample (urine)
- Extract DNA from sample (sometimes remove human DNA)
- Then sequence DNA using a particular method
- Once DNA sequenced you analyse it using computational tools
38
Q
Genomic diagnostic vs conventional diagnostic
A
Genomic diagnostics allow for higher resolutions than conventional diagnostics e.g. allows you to identify individual species of a type of bacteria whilst conventional can not.
